Disclosed herein are methods for assembling DNA molecules in a predetermined order in a DNA construct and methods of using such constructs.
Many methods have been developed and used for assembling DNA molecules. Examples of these are cloning by restriction endonuclease digestion followed by ligation of compatible ends, T-A cloning directly from a polymerase chain reaction product, recombination-based cloning and ligation-independent cloning of polymerase chain reaction products.
Ligation-independent cloning is a highly efficient and cost effective method for assembling DNA molecules using non-covalent, bi-molecular association of terminal, single-stranded DNA segments to assemble DNA molecules. Terminal, single-stranded DNA segments can be incorporated into DNA molecules by a variety of methods. Examples of methods for producing terminal, single-stranded DNA segments include, but are not limited to, (a) ligation of adapter molecules containing a terminal, single-stranded DNA segment, (b) incorporation of nicking endonuclease sites during polymerase chain reaction, (c) exonuclease digestion and (d) incorporation of deoxy-uridine residues during polymerase chain reaction which can be deaminated by the enzyme, uracil-N glycosylase. Such methods are described in Aslanidis and de Jong, Nucleic Acids Research, 18 (20), pages 6069-6074 (1990); U.S. Pat. No. 5,580,759 (Yang, et al.); U.S. Pat. No. 5,137,814 (Rashtchian et al.) and U.S. Pat. No. 6,495,318 (Harney), all of which are incorporated herein by reference. Once assembled, the DNA construct is repaired and replicated in the bacterial host using the host's endogenous systems.
Conventional cloning methods can be time consuming and often rely on a series of sub cloning steps to produce the final DNA construct. DNA constructs used in plant transformation contain a number of necessary components for replication, transformation and selection. For example, the DNA construct must have an origin of replication and selectable marker for the bacterial hosts, both for Escherichia coli as well as Agrobacterium tumefaciens. For plant transformation, the construct must also contain a right and left border sequence to allow for the transfer and stable incorporation of the DNA from Agrobacterium to the plant host cells. The DNA construct will also usually contain a eukaryotic promoter, which drives the expression of protein coding sequences and transcriptional terminators. DNA constructs typically also comprise selectable or screenable markers used in identifying the transformed plant host cells. The final DNA construct can be quite large. Therefore, a need exists for developing simpler and widely applicable methods for producing constructs used in plant transformation.